Program Director/Principal Investigator (Last, First, Middle): Hase, Claudia C. 1R21 AI064190-01A2 All living cells establish transmembrane electrochemical gradients with the help of primary ion pumps. Primary Na+ pumps have been discovered in many microorganisms and a transmembrane circulation of Na+ ions may play a significant role in the physiology of several pathogenic bacteria. Pathogenic bacteria are exposed to a wide range of environments both within and outside the host and have to be able to adapt to changes in the surrounding tissue to establish an infection. Although little is known about the role of the sodium cycle of energy during infection by Yersinia pestis, the presence of several sodium-dependent systems in the genome of this organism strongly suggests that this organism relies on sodium bioenergetics for at least part of its energy metabolism. We have previously hypothesized that bacterial sodium pumps involved in pH and sodium homeostasis are important for bacterial virulence and thus should provide a target for the development of a novel intervention strategy. Indeed, our preliminary results demonstrated a dramatic loss of virulence in the main sodium/proton antiporter (NhaA) mutant of Y. pestis in an animal infection model. The objectives of this application are to further define the contribution of sodium bioenergetics to Y. pestis physiology and virulence in different experimental assays. We will examine the roles of the different sodium pumps in the membrane energetics of Y. pestis by combining extensive genetic manipulations with various bioenergetic measurements. Furthermore, we will analyze the virulence traits of these mutants in vitro and in vivo to better understand the importance of these sodium pumps during the different stages of infection. Sodium bioenergetics probably plays a role in all of the various phases of the Y. pestis life cycle. Thus, the outcome of the proposed research will be of value to understanding fundamental biological concepts related to Y. pestis ecology in the lumen of its different hosts. It therefore offers great potential for application and will be of interest to a wide audience within the research community.